This invention is related to the genetic engineering of plants and to a means and method for conferring a plurality of traits, including resistance to viruses, to a plant using a vector encoding a plurality of genes, such as coat protein genes, protease genes, or replicase genes.
Many agriculturally important crops are susceptible to infection by plant viruses, which can seriously damage a crop, reduce its economic value to the grower, and increase its cost to the consumer. Attempts to control or prevent infection of a crop by a plant virus have been made, yet viral pathogens continue to be a significant problem in agriculture.
Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques. Such an approach is advantageous in that the genetic material which provides the protection is incorporated into the genome of the plant itself and can be passed on to its progeny. A host plant is resistant if it possesses the ability to suppress or retard the multiplication of a virus, or the development of pathogenic symptoms. xe2x80x9cResistantxe2x80x9d is the opposite of xe2x80x9csusceptible,xe2x80x9d and may be divided into: (1) high, (2) moderate, or (3) low resistance, depending upon its effectiveness. Essentially, a resistant plant shows reduced or no symptom expression, and virus multiplication within it is reduced or negligible. Several different types of host resistance to viruses are recognized. The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.
Potyviruses are a distinct group of plant viruses which are pathogenic to various crops, and which demonstrate cross-infectivity between plant members of different families. Potyviruses include watermelon mosaic virus-2 (WMV-2); papaya ringspot virus strains papaya ringspot and watermelon mosaic I (PRV-p and PRV-w), two closely related members of the plant potyvirus group which were at one time classified as distinct virus types, but are presently classified as different strains of the same virus; zucchini yellow mosaic virus (ZYMV); potato virus Y; tobacco etch and many others. For example, see Table I of published European patent application 578,627.
These viruses consist of flexous, filamentous particles of dimensions approximately 780xc3x9712 nanometers. The viral particles contain a single-stranded RNA genome containing about 10,000 nucleotides of positive (+, coding, or sense) polarity. Translation of the RNA genome of potyviruses shows that the RNA encodes a single large polyprotein of about 330 kD. This polyprotein contains several proteins, one of which is a 49 kD protease that is specific for the cleavage of the polyprotein into at least six (6) other peptides. These proteins can be found in the infected plant cell and form the necessary components for viral replication. One of the proteins contained within this polyprotein is a 35 kD capsid or coat protein which coats and protects the viral RNA from degradation. Another protein is the nuclear inclusion protein, also referred to as replicase, which is believed to function in the replication of the viral RNA. In the course of a potyviral infection, the replicase protein (60 kDa, also referred to as the nuclear inclusion B protein) and the protease protein (50 kDa, also referred to as the nuclear inclusion I or nuclear inclusion A protein) are posttranslationally transported across the nuclear membrane into the nucleus of the plant cell at the later stages of viral infection and accumulate to high levels.
Generally, the coat protein gene is located at the 3xe2x80x2-end of the RNA, just prior to a stretch of terminal adenine nucleotide residues (200 to 300 bases). The location of the 49 Kd protease gene appears to be conserved in these viruses. In the tobacco etch virus, the protease cleavage site has been determined to be the dipeptide Gln-Ser, Gln-Gly or Gln-Ala. Conservation of these dipeptides as the cleavage sites in these viral polyproteins is apparent from the sequences of the above-listed potyviruses.
Expression of the coat protein genes from tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, and potato virus X, among others, in transgenic plants has resulted in plants which are resistant to infection by the respective virus. Some evidence of heterologous protection has also been reported. For example, Namba et al., Phytopathology, 82, 940 (1992) report that expression of coat protein genes from watermelon mosaic virus-2 or zucchini yellow mosaic virus in transgenic tobacco plants conferred protection against six other potyviruses: bean yellow mosaic virus, potato virus Y, pea mosaic virus, clover yellow vein virus, pepper mottle virus and tobacco etch virus. Stark et al., Biotechnology, 1, 1257 (1989) report that expression of the potyvirus soybean mosaic virus in transgenic plants provided protection against two serologically unrelated potyviruses: tobacco etch virus and potato virus Y.
However, expression of a preselected coat protein gene does not reliably confer heterologous protection to a plant. For example, transgenic squash plants containing the CMV-C coat protein gene and which have been shown to be resistant to CMV-C strain, are not protected against several highly virulent strains of CMV, including CMV-V-27 and CARNA-5. Thus, a need exists for improved methods to impart potyvirus resistance to plants.
The present invention provides a recombinant chimeric DNA molecule comprising a plurality of DNA sequences each of which comprises a promoter operably linked to a DNA sequence which encodes a virus-associated protein, such as a coat protein (cp), a protease, or a replicase, wherein said DNA sequences are expressed in virus-susceptible plant cells transformed with said recombinant DNA molecule to impart resistance to infection by each of said viruses. Preferably, the DNA sequences are linked in tandem, i.e., exist in head to tail orientation relative to one another. Also, preferably substantially equal levels of resistance to infection by each of said viruses occurs in plant cells transformed with said plurality of DNA sequences.
Preferably, each DNA sequence is also linked to a 3xe2x80x2 non-translated DNA sequence which functions in plant cells to cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3xe2x80x2 end of the transcribed mRNA sequences. Preferably, the virus is a plant-associated virus, such as a potyvirus.
Thus, the present DNA molecule can be employed as a chimeric recombinant xe2x80x9cexpression construct,xe2x80x9d or xe2x80x9cexpression cassettexe2x80x9d to prepare transgenic plants that exhibit increased resistance to infection by at least two plant viruses, such as potyviruses. The present cassettes also preferably comprise at least one selectable marker gene or reporter gene which is stably integrated into the genome of the transformed plant cells in association with the viral genes. The selectable marker and/or reporter genes facilitate identification of transformed plant cells and plants. Preferably, the virus gene array is flanked by two or more selectable marker genes, reporter genes or a combination thereof. Another aspect of the present invention is a method of preparing a virus-resistant plant, such as a dicot, comprising:
(a) transforming plant cells with a chimeric recombinant DNA molecule comprising a plurality of DNA sequences, each comprising a promoter functional in said plant cells, operably linked to a DNA sequence, which encodes a protein associated with a virus which is capable of infecting said plant;
(b) regenerating said plant cells to provide a differentiated plant;
and
(c) identifying a transformed plant which expresses the DNA sequences so as to render the plant resistant to infection by said viruses, preferably at substantially equal levels of resistance to infection by each virus.
Yet another object of the present invention is to provide a method for providing resistance to infection by viruses in a susceptible Cucurbitaceae plant which comprises:
(a) transforming Cucurbitaceae plant cells with a DNA molecule encoding a plurality of proteins from viruses which are capable of infecting said Cucurbitaceae plant;
(b) regenerating said plant cells to provide a differentiated plant;
and
(c) selecting a transformed Cucurbitaceae which expresses the virus proteins at levels sufficient to render the plant resistant to infection by said viruses.
It is a further object of the present invention to provide multi-virus resistant transformed plant which contains stably-integrated DNA sequences encoding virus proteins.
It is still a further object of the present invention to provide virus resistant transformed plant cells which contain a plurality of viral genes, i.e., 2-7 or more genes, which are expressed as virus proteins from the same virus strain, from different virus strains as from different members of the virus group, such as the potyvirus group.
The present invention is exemplified primarily by the insertion of multiple virus cp expression cassettes into a binary plasmid and subsequent characterization of resulting plasmids. Combinations of CMV, ZYMV, WMV-2, SQMV, and PRV coat protein expression cassettes were placed in the binary plasmid pPRBN. Subsequently, binary plasmids harboring multiple cp expression cassettes were mobilized into Agrobacterium for use in plant transformation procedures. Binary plasmids harboring multiple expression cassettes are employed to transfer two or more virus coat protein transformation-susceptible genes into plants, such as members of the Cucurbitaceae family, along with the associated selectable marker and/or reporter genes.
Thus, the present invention provides a genetic engineering methodology by which multiple traits can be manipulated and tracked as a single gene insert, i.e., as a construct which acts as a single gene which segregates as a single Mendelian locus. Although the invention is exemplified via virus resistance genes, in practice, any combination of genes could be linked. Therefore one could track a block of genes that provide traits such as disease resistance, plus enhanced herbicide resistance, plus extended shelf life, and the like, by simply tracking the linked selectable marker or reporter gene which has been incorporated into the transformation vector.
It was also discovered that when multiple tandem genes are inserted, they preferably all exhibit substantially the same degrees of efficacy, and more preferably substantially equal degrees of efficacy, wherein the term xe2x80x9csubstantialxe2x80x9d as it relates to viral resistance is defined with reference to the assays described in the examples hereinbelow. For example, if one examines numerous transgenic lines containing an intact ZYMV and WMV-2 coat protein insert, one finds that if a line is immune to infection by ZYMV it is also immune to infection by WMV-2. Similarly, if a line exhibits a delay in symptom development to ZYMV it will also exhibit a delay in symptom development to WMV2. Finally, if a line is susceptible to ZYMV it will be susceptible to WMV-2. This phenomenon is unexpected. If there were not a correlation between the efficacy of each gene in these multiple gene constructs this approach as a tool in plant breeding would probably be prohibitively difficult to use. Even with single gene constructs, one must test numerous transgenic plant lines to find one that displays the appropriate level of efficacy. The probability of finding a line with useful levels of expression can range from 10-50% (depending on the species involved).
If the efficacy of individual genes in a Ti plasmid containing multiple genes were independent, the probability of finding a transgenic line that was resistant to each targeted virus would decrease dramatically. For example, in a species in which there is a 10% probability of identifying a line with resistance using a single gene insert, is transformed with a triple-gene construct CZW and each gene display an independent levels of efficacy, the probability of finding a line with resistance to CMV, ZYMV and WMV-2 would be 0.1xc3x970.1xc3x970.1=0.001 or 0.1%. However, since the efficacy of multivalent genes is not independent of each other the probability of finding a line with resistance to CMV, ZYMV and WMV-2 is still 10% rather than 0.1%. Obviously this advantage becomes more pronounced as constructs containing four or more genes are used.